Assays are known that take advantage of a sensitive and highly specific interaction between cuprous (Cu.sup.+1) ions and the sodium salt of bicinchoninic acid (BCA) for the determination of protein concentrations in solution. See Smith, et al., "Measurement of Protein Using Bicinchoninic Acid", Analyt. Biochem. 150:76-85 (1985). At alkaline pH, proteins will reduce Cu.sup.+2 to Cu.sup.-1 in the presence of BCA, and this reaction forms product that absorbs light strongly at a wavelength of 562 nm. Since the production of Cu.sup.+1 in the BCA assay is a function of protein concentration and incubation time, the protein content of unknown samples may be determined spectrophotometrically by comparing the sample absorption spectrum to that of known protein standards.
The ease with which an accurate BCA assay may be performed has made it preferable to and of greater general utility than the Biuret, Lowry or Bradford techniques reported in the prior art. See Lowry, et al. "Protein Measurement with the Folin Phenol Reagent",J. Biol. Chem. 193:265-275 (1951); "DC Protein Instruction Manual" Bio-Rad Laboratories, Richmond, Calif. (modified Lowry Assay); and Bradford, "A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein utilizing the Principle of Protein-Dye Binding", Analyt. Biochem. 72:248-254 (1976). This is especially true because of the low levels of interference caused by reagents commonly used in protein preparations, such as detergents, certain buffers and salts, and some reducing agents. See Pierce, "BCA* Protein Assay Reagent Protocol and Information Manual 23220/23225" Pierce Chemical Co., Rockford, Ill. (1989); Smith, et al. "Measurement of Protein Using Bicinchoninic Acid", Analyt. Biochem. 150:76-85 (1985).
A particular concern in the use of protein assays involves instances where such interfering compounds accumulate or partition differentially among samples. For example, some detergents may associate preferentially with membrane proteins compared to cytosolic proteins during chromatographic purification. In such situations, artificially high protein levels are determined for membrane proteins, even if appropriate corrections are made to account for the concentration of detergent in the buffer. The use of the BCA assay obviates this problem since many detergents do not interfere with the assay. See Smith, et al., referenced above.
Kits taking advantage of the above-described BCA reaction are available, for example, from Pierce Chemical Co. (Rockford, Ill.). Three standard variations of the BCA based protein assay are suggested by Pierce with incubation at either room temperature, 37 C., or 60 C. for a period of 30 to 120 minutes. See Pierce, "BCA* Protein Assay Reagent Protocol and Information Manual 23220/23225" Pierce Chemical Co., Rockford, Ill., (1989). In general, it is known that the sensitivity of standard BCA assays increases with elevated incubation temperature and/or longer incubation time, and the assay can be easily adjusted to the range of interest. However, the time required for BCA protein analysis diminishes its utility for a number of reasons. For example, a large number of samples cannot be examined in a reasonable amount of time, nor can samples be assayed from a system undergoing a changes on the order of minutes, since the assay results would always lag the changes in the system. Additionally, the presently available assays are disruptive in procedures that require protein determination at multiple intermediate steps. It would therefore be desirable to reduce the incubation time of protein assays without diminishing the sensitivity or accuracy of the assay. It is therefore an object of the present invention to provide methods and apparatus for determining protein concentration based on known assays that may be performed using incubation times on the order of seconds, while retaining appropriate levels of sensitivity, reliability, accuracy and resistance to interfering compounds. It is a further object of the present invention to provide assays that are easy to use and easily integrated into current laboratory practice.